Organelle movement through cytoplasm, which occurs in virtually all eukaryotic cells, plays an important role in transporting lipids and proteins between regions or compartments of the cell. Organelle transport was described ober a century ago, yet the mechanism responsible for generating this movement has remained a mystery, although it is now clear that microtubules and microtubule-based motility proteins are involved in this process. Using neurons as model systems for examining this important phenomenon, my research approach is to reconstitute microtubule- based transport in vitro using isolated components that can then be biochemically analyzed. The overall goals if this proposal are to identify the molecules that comprise the force generating machinery that powers organelle transport, to determine how these proteins generate movement and to understand how motility is regulated. The specific aims are: 1) to study the biochemistry and in vitro motility of a newly described force generating protein termed kinesin, distinct from myosin and dynein, that appears to mediate anterograde movement of organelles in axons. Research will focus upon investigating the molecular interaction of kinesin with microtubules and nucleotides, the mechanism and regulation of force production, the association of kinesin with organelles, and the characterization of functional domains of kinesin. 2) to identify, purify and characterize a second motility protein in crude axoplasmic supernatants that generates movement along microtubules in the opposite direction to kinesin. This protein may be involved in the retrograde transport of organelles. 3) to ascertain how kinesin and the retrograde motor are involved in transporting organelles by reconstituting movement using purified proteins and organelles and to identify signals that control the direction of transport of organelles along microtubules. The in vitro motility assays, the ability to prepare sufficient quantities of kinesin for biochemical characterization and the availability of specific antibodies against kinesin have opened up new possibilities for understanding organelle transport and other forms of microtubule-based motility at a molecular level. The studies proposed here address fundamental problems in modern cell biology and contribute to an understanding of transport processes that are medically relevant such as hormone secretion and nerve regeneration.